Project Summary The worldwide prevalence of obesity has reached pandemic proportions. Obesity has strong inflammatory underpinnings, which are associated with the development of type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH). However, the mechanisms by which obesity provokes aberrant inflammation are not clearly defined. In this study, we aim to demonstrate that in obesity, the mTOR pathway enhances the biogenesis and secretion of pro-inflammatory extracellular vesicles (EVs) carrying a distinct set of extracellular RNAs (exRNAs) from hepatocytes and that these hepatocyte-derived EVs (H-EVs) cause the aberrant inflammation. EVs, including exosomes (30?150 nm) and microvesicles (100?1000 nm), are released from many cell types into the extracellular space and are distributed in body fluids. These EVs are taken up by neighboring or distant cells and subsequently modulate functions of recipient cells. Using novel computational methods9, we identified strong associations between human genetic variations of genes regulating EV biogenesis and metabolic syndrome, particularly T2D. Our analyses of EVs from adolescent obese patients undergoing bariatric surgery have shown that serum EV concentration is inversely correlated to metabolic improvements in insulin sensitivity and inflammation post-surgery, with unique EVs? exRNA profiles. Further, our newly established mouse model that permits monitoring of specific cell type-derived EVs in vivo indicates that in obesity, H-EVs behave like a pathogen recognized by macrophages and induce inflammation. Mechanistically, we found that in hepatocytes, the insulin-mTOR pathway enhances secretion of EVs and that insulin- stimulated EVs are more pro-inflammatory in activating macrophages. We have also discovered that a component of the RNA silencing machinery is a mediator of the pro-inflammatory EVs and is required for the EV-induced macrophage activation. These preliminary data suggest unique and pivotal roles for H-EVs, leading us to hypothesize that during the development of hyperinsulinemia, hepatocytes secrete pathologic EVs carrying unique exRNAs which are sensed by recipient macrophages, promoting aberrant inflammation. Studies in this proposal will: (1) define the molecular pathway that confers the proinflammatory trait to the H- EVs, (2) determine the role of RNA silencing machinery in macrophages as a mediator of pro-inflammatory EVs, and (3) evaluate the role of EVs in immunometabolism in human obese patients. By utilizing our novel mouse models coupled with access to human samples, our systematical approaches will demonstrate novel mechanisms concerning how the pathogenicity of H-EVs is involved in the development of inflammation in hyperinsulinemia and obesity.